ABSTRACT T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes that affects thousands of children and adults annually in the United States. Despite improved outcomes for patients with primary T- ALL, the elevated cure rates are largely attributable to highly toxic chemotherapy with both short- and long- term adverse effects. Moreover, chemotherapy is often ineffective against relapsed T-ALL, which has a dismal 5-year survival rate of 30% in children and <10% in adults. Thus, identification of new actionable drug targets in T-ALL is a major research and clinical imperative. The overall objective of this work is to define the role of a protein tyrosine phosphatase, PRL-3, in T-ALL growth and self-renewal. PRL3 is genomically amplified with the MYC oncoprotein in 12% of human T-ALL and is highly expressed in 45% of primary T-ALL. The central hypothesis is that oncogenic PRL3 drives T-ALL growth by increasing self-renewal of relapse-driving leukemia propagating cells (LPCs) and suppressing apoptosis, and that its blockade kills T-ALL cells. The rationale for the proposed research is that by understanding how PRL3 promotes T-ALL growth and progression, drugs targeting this phosphatase or its immediate downstream targets could be developed for the treatment of T-ALL. Aim 1 will identify the cellular function by which PRL3 modulates T-ALL growth, maintenance and self-renewal. For these studies, an innovative zebrafish transgenic model of MYC-induced T-ALL will be used to assess a role for PRL3 in altering T-ALL initiation, heterogeneity, self-renewal and apoptosis. This work will be extended to human patient-derived xenograft models, establishing PRL3 as a bona fide drug target in human T-ALL. The working hypothesis underlying Aim 1 is that PRL3 enhances LPC self-renewal and suppresses apoptosis. It is also expected that PRL3 will be required for continued leukemia growth in vivo. Aim 2 will uncover the molecular mechanism(s) by which PRL3 regulates the growth of human T-ALL. We will use a novel phosphoproteomic approach, a CRISPR/Cas9 screen and phosphatase substrate trap assay to identify candidate substrates that regulate processes that induce apoptosis. Detailed molecular analysis will identify the precise mechanism by which PRL3 substrates alter the viability and growth of human T-ALL. The working hypothesis for Aim 2 is that direct substrates of PRL3 suppress downstream apoptotic pathways in human T- ALL. Successful completion of this work will 1) identify actionable drug targets downstream of the PRL3 pathway in human T-ALL, and 2) discover the downstream substrates of PRL3, likely providing new biomarkers for assessing drug effects on T-ALL and additional drug targets for the treatment of T-ALL. Our work is significant and will likely have a positive translational impact because it provides novel mechanistic insights into how PRL3 functions in T-ALL and establishes this phosphatase as a bona fide therapeutic target in T-ALL. The oncogene status of PRL3 in a wide range of cancers suggests that our studies will have a positive translational impact on tumors other than T-ALL that are addicted to PRL3 oncogene expression.